The present invention relates to a novel peptide which has endothelin-antagonizing activity, and an intermediate for its synthesis. The peptide has excellent endothelin-antagonizing activity, and is therefore useful for treatment of hypertension, asthma, cerebral apoplexy, angina pectoris, acute renal failure, cardiac infarction, cerebral vasospasm, etc.
Endothelin is a cyclic peptide which possesses a strong, long-lasting vasoconstricting effect, and is thought to be one of the substances responsible for hypertension, asthma, acute renal failure, cardiac infarction, cerebral apoplexy, angina pectoris and cerebral vasospasm. Consequently, a substance which antagonizes endothelin and inhibits its effects is expected to be useful for the treatment and prevention of these diseases.
It is known that the cyclic peptide represented by the formula (A): 
(wherein U represents D-Val or D-allo-Ile, R1 represents hydrogen or an amino-protective group, and R2 represents hydrogen or a carboxyl-protective group) exhibits the endothelin antagonism (Japanese Published Unexamined Patent Application No. 130299/91).
The present inventors have made a screening of numerous substances produced by microorganisms in order to find a naturally occurring physiologically active substance having endothelin-antagonizing activity, have succeeded in isolating substances from the cultures of microorganisms belonging to the genus Streptomyces newly isolated from the soil, which substances have endothelin-antagonizing activity and a suppressing effect on the increase in intracellular calcium and intracellular guanosine-3xe2x80x2,5xe2x80x2-cyclic monophosphate concentrations due to endothelin, and have named the substances Compound (I-1), Compound (I-2) and Compound (I-3). As the result of further investigations, the present inventors have successfully determined the structure of these substances, have synthesized novel derivatives of these substances, and thus have completed the present invention.
According to the present invention, there is provided a peptide compound represented by the following formula (I):
X-A-Trp-B-Gly-Thr-E-G-Y xe2x80x83xe2x80x83(I) SEQ ID NO. 2
wherein
A represents Asn or Asp;
B represents His or Lys;
E represents Ala or Ser;
G represents Ala or Pro;
X represents 
Y represents hydroxy, lower alkoxy, amino, 
wherein
each of X1 and X3 represents hydrogen, benzyloxycarbonyl, t-butyloxycarbonyl or 9-fluorenylmethyloxycarbonyl, or carbonyl-substituted or unsubstituted lower alkanoyl;
each of X2 and Y2 represents hydrogen;
Y1 represents hydroxy, lower alkoxy or amino; or
X1 and Y1, or X2 and Y2 are combined together to form a single bond as X1xe2x80x94Y1 or X2xe2x80x94Y2; and
Z represents hydroxy, lower alkoxy, benzyloxy, benzhydryloxy, amino, 
where
Z2 is hydroxy, lower alkoxy, benzyloxy, benzhydryloxy or amino,
Glyxe2x80x94Z1 
where
Z1 is hydroxy, lower alkoxy, benzyloxy, benzhydryloxy, amino, 
where Z2 is as defined previously, or Z1 is combined with X1 to form a single bond as X1-Z1,
Alaxe2x80x94Z1 
where Z1 is as defined previously,
Valxe2x80x94Z1 
where Z1 is as defined previously,
Trpxe2x80x94Z1 
where Z1 is as defined previously,
Trpxe2x80x94Glyxe2x80x94Z1 
where Z1 is as defined previously,
Trp-Asn-Tyr-Tyr-Trp-Z1 
where Z1 is as defined previously,
xe2x80x83Trp-Phe-Phe-Asn-Tyr-Tyr-7Hyt-Z1, xe2x80x83xe2x80x83SEQ ID NO: 4
where Z1 is as defined previously, and 7Hyt represents 7-hydroxytryptophan,
Trp-Ile-Ile-Tr-Z1, xe2x80x83xe2x80x83SEQ ID NO: 5 
where Z1 is as defined previously,
Trp-Val-Tyr-Phe-W-His-Leu-Asp-Ile-Ile-Trp-Z1, xe2x80x83xe2x80x83SEQ ID NO: 6 
where Z1 is as defined previously and W represents Ala, Ser or Cys,
Trp-W-His-Leu-Asp-Ile-Ile-Trp-Z1, xe2x80x83xe2x80x83SEQ ID NO: 7 
where Z1 and W are as defined previously,
Trp-Val-Tyr-Tyr-W-His-Leu-Asp-Ile-Ile-Trp-Z1, xe2x80x83xe2x80x83SEQ ID NO: 8 
where Z1 and W are as defined previously,
Trp-Leu-Tyr-Phe-W-His-Gln-Asp-Val-Ile-Trp-Z1, xe2x80x83xe2x80x83SEQ ID NO: 9 
where Z1 and W are as defined previously,
Trp-Val-Tyr-Phe-W-Phe-Phe-Asn-Tyr-Tyr-Trp-Z1, xe2x80x83xe2x80x83SEQ ID NO: 10 
where Z1 and W are as defined previously,
Trp-Phe-Phe-Asn-Tyr-Tyr-W-His-Leu-Asp-Ile-Ile-Trp-Z1, xe2x80x83xe2x80x83SEQ ID NO: 11 
where Z1 is as defined previously,
Trp-Phe-Phe-Asn-Tyr-Tyr-Asn-Ile-Ile-Trp-Z1, xe2x80x83xe2x80x83SEQ ID NO: 12 
where Z1 is as defined previously,
J-Phe-M-Q-Tyr-R-T-Z1, xe2x80x83xe2x80x83SEQ ID NO: 13 
where
J is Trp or a single bond,
M is Phe or a single bond,
Q is Asn or a single bond,
R is Tyr or a single bond,
T is
Trp,
Ala,
Phe,
Tyr,
Trp-Trp,
Asn-Tyr-Tyr-Trp, xe2x80x83xe2x80x83SEQ ID NO: 14 
Trp-Asn-Tyr-Tyr-Trp, xe2x80x83xe2x80x83SEQ ID NO: 15 
Try-Val-Tyr-Phe-W-His-Leu-Asp-Ile-Ile-Trp, xe2x80x83xe2x80x83SEQ ID NO: 16 
where W is as defined previously, or a single bond,
2 or more of J, M, Q, R and T are not a single bond simultaneously, and Z1 is as defined previously, or a pharmaceutically acceptable salt thereof.
Also, according to the present invention there is provided an intermediate represented by the following formula (II):
X4-J-Phe-M-Q-Tyr-R-T-Z3 xe2x80x83xe2x80x83(II) SEQ ID NO: 17 
wherein X4 represents hydrogen or benzyloxycarbolnyl, t-butyloxycarbonyl or 9-fluorenylmethyloxycarbonyl, Z3 represents hydroxy, lower alkoxy, benzyloxy or benzhydryloxy, and J, M, Q, R and T are as defined previously, which is useful for the synthesis of a peptide compound represented by the formula (I).
The peptide compound represented by the above formula (I) is referred to as Compound (I), and compounds represented by the other formulas are similarly referred to.
In the definitions for the above formulas (I) and (II), the lower alkyl and the alkyl moiety in lower alkoxy mean linear or branched alkyl having 1-6 carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, neopentyl, hexyl and isohexyl. The lower alkanoyl means those having linear or branched 1-6 carbon atoms, such as formyl, acetyl, propionyl, butyryl, valeryl, pivaloyl and pentanoyl.
As the pharmaceutically acceptable salt of Compound (I), mention may be made of an acid addition salt, a metal salt and an organic base addition salt. Specifically, the pharmaceutically acceptable acid addition salt includes, for example, inorganic acid salts such as hydrochloride, sulfate, phosphate, etc. and organic acid salts such as acetate, maleate, fumarate, tartrate, citrate, etc. The pharmaceutically acceptable metal salt includes, for example, alkali metal salts such as sodium salt, potassium salt, etc., alkaline earth metal salts such as magnesium salt, calcium salt, etc., aluminum salt, zinc salt, etc. The pharmaceutically acceptable organic base addition salt includes, for example, primary amines such as methylamine, ethylamine, aniline, etc., secondary amines such as dimethylamine, diethylamine, pyrrolidine, piperidine, morphopline, piperazine, etc., tertiary amines such as trimethylamine, triethylamine, N,N-dimethylaniline, pyridine, etc. and ammonia, etc.
The method for producing compounds (I) and (II) is given below.
Of Compound (I), Compound (I-1), Compound (I-2) or Compound (I-3) having the structures listed below is produced by culturing in a medium a microorganism belonging to the genus Streptomyces capable of producing Compound (I-1), Compound (I-2) or Compound (I-3) to form and accumulate Compound (I-1), Compound (I-2) or Compound (I-3) in the culture, and recovering Compound (I-1), Compound (I-2) or Compound (I-3) therefrom. 
The specific preferred microorganism is an actinomyces such as Streptomyces sp. RE-701 which was isolated by the present inventors from the soil in the areas of Kitashitara-gun, Aichi; and Streptomyces sp. RE-629 which was isolated by the present inventors from the soil in the areas of Tsuno-gun, Yamaguchi.
The bacteriological properties of Streptomyces sp. RE-701 strain are described below.
I. Morphology
In a usual agar medium, the strain RE-701 possesses a septum and forms branched aerial mycelia and substrate mycelia. Characteristic fragmentation of the substrate mycelia is not observed. Also, there is no formation of sporangia or sclerotia.
Chains of more than 10 spores form as the end of sporophores simply branched from the aerial mycelium, and the shapes are open loops or spirals. The mature spores are oval of size 0.4-0.5 xcexcmxc3x970.6-0.8 xcexcm with a smooth surface and without flagella.
II. Growth conditions on various media
The strain RE-701 grows normally or vigorously, forming grayish aerial mycelia on usual synthetic and natural media. The substrate mycelia exhibit a grayish-white to brown color. Soluble pigments are not produced.
The growth and color characteristics of the strain RE-701 when cultured in various media at 28xc2x0 C. for 10 days are shown below. The color indications follow the classifications in the Color Harmony Manual, Container Corporation of America.
1. Glucose-asparagine agar medium
xe2x80x83Growth, reverse side color: moderate, putty (1xc2xd ec)
xe2x80x83Aerial mycelia: moderate, white (a)
xe2x80x83Soluble pigment: none
2. Glycerol-asparagine agar medium
xe2x80x83Growth, reverse side color: Poor, oyster white (b)
xe2x80x83Aerial mycelia: somewhat poor, white (a)
xe2x80x83Soluble pigment: none
3. Sucrose-nitrate agar medium
xe2x80x83Growth, reverse side color: good, putty (1xc2xd ec)
xe2x80x83Aerial mycelia: moderate, gray (f)
xe2x80x83Soluble pigment: none
4. Starch-inorganic salt agar medium
xe2x80x83Growth, reverse side color: good, light antique gold (1xc2xd ic)
xe2x80x83Aerial mycelia: abundant, gray (g)
xe2x80x83Soluble pigment: none
5. Tyrosine agar medium
xe2x80x83Growth, reverse side color: poor, light tan (3 gc)
xe2x80x83Aerial mycelia: poor, light beige (3 ec)
xe2x80x83Soluble pigment: none
6. Nutrient agar medium
xe2x80x83Growth, reverse side color: moderate, honey gold (2 ic)
xe2x80x83Aerial mycelia: somewhat poor, white (a)
xe2x80x83Soluble pigment: none
7. Malt extract-yeast extract agar medium
xe2x80x83Growth, reverse side color: good, mustard gold (2 ne)
xe2x80x83Aerial mycelia; moderate, white (a)
xe2x80x83Soluble pigment: none
8. Oatmeal agar medium
xe2x80x83Growth, reverse side color: somewhat good, white (1xc2xd lg)
xe2x80x83Aerial mycelia: poor, charcoal gray (o)
xe2x80x83Soluble pigment: none
III. Physiological properties
The physiological properties of the strain RE-701 are described below. The growth temperature range indicates the results obtained by observation for 6 days. With respect to the other items, the results obtained by observation for 2 weeks at 28xc2x0 C. are shown.
(1) Carbon utilization: A Pridham and Gottlieb inorganic medium (ISP No. 9) was used as a basal medium.
The strain RE-701 assimilates D-glucose, D-fructose, sucrose, inositol, raffinose and D-mannitol, but do not assimilate D-arabinose and L-rhamnose. The assimilation of D-xylose is uncertain.
(2) Effects on milk: coagulation, no liquefaction
(3) Hydrolysis of starch: positive
(4) Growth temperature range: 7-41xc2x0 C.
(5) Formation of melanoid pigments: negative
(6) Liquefaction of gelatin: negative
IV. Cell wall composition
In analysis for diaminopimelic acid by hydrolysis of the whole cells, only LL-diaminopimelic acid was detected.
The strain is classified in the genus Streptomyces of Actinomycetales, on the basis of the spore chains"" formation on the aerial mycelia and the configuration of diaminopimelic acid, etc.
Therefore, the strain was named as Streptomyces sp. RE-701, and has been deposited with the Fermentatipn Research Institute, Agency of Industrial Science and Technology 1-3, Higashi 1 chome Tsukuba-shi Ibaraki-ken 305, Japan, under FERM BP-3624, as of Oct. 29, 1991. The deposited strain has been accepted for deposit under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purpose of Patent Procedure. All restrictions on the availability to the public of the deposited strain will be irrevocably removed upon the issuance of a patent.
The bacteriological characteristics of Streptomyces sp. RE-629 are described below.
I. Morphology
In a usual agar medium, the strain RE-629 possesses a septum and form branched aerial mycelia and substrate mycelia. Characteristic fragmentation of the substrate mycelia is not observed. Also, there is no formation of sporangia or sclerotia.
Chains of more than 10 spores form at the end of sporophores simply branched from the aerial mycelium, and the shapes are open loops or spirals. The mature spores are oval of size 0.7-0.8 xcexcmxc3x970.7-1.0 xcexcm with a smooth surface and without flagella.
II. Growth conditions on various media
The strain RE-629 grows normally or vigorously, forming grayish aerial mycelia on usual synthetic and natural media. The substrate mycelia exhibit a grayish-white to brown color. Brownish soluble pigments are produced on specific media.
The growth and color characteristics of the strain RE-629 on various media at 28xc2x0 C. after 14 days are shown below. The color indications follow the classifications in the Color Harmony Manual, Container Corporation of America.
1. Glycerol-asparagine agar medium
xe2x80x83Growth, reverse side color: good, bamboo (2 gc)
xe2x80x83Aerial mycelia; good, natural (2 dc)
xe2x80x83Soluble pigment: none
2. Starch-inorganic salt agar medium
xe2x80x83Growth, reverse side color: good, dull gold (2 ng)
xe2x80x83Aerial mycelia: abundant, silver gray (3 fe)
xe2x80x83Soluble pigment: present
3. Malt extract-yeast extract agar medium
xe2x80x83Growth, reverse side color: Good, oak brown (4 pi)
xe2x80x83Aerial mycelia: good, silver gray (3 fe)
xe2x80x83Soluble pigment: present
4. Oatmeal agar medium
xe2x80x83Growth, reverse side color: moderate, olive (1xc2xd pl)
xe2x80x83Aerial mycelia: moderate, lamp black (o)
xe2x80x83Soluble pigment: present
III. Physiological properties
The physiological properties of the strain RE-629 are described below. The growth temperature range indicates the results obtained by observation for 5 days. With respect to the other items, the results obtained by observation for 2 weeks at 28xc2x0 C. are shown.
(1) Carbon utilization: A Pridham and Gottlieb inorganic medium (ISP No. 9) was used as the basal medium. The strain RE-629 assimilates D-glucose, D-fructose, sucrose, inositol, raffinose, D-mannitol and D-xylose, but does not assimilate L-rhamnose. The assimilation of D-arabinose is uncertain.
(2) Growth temperature range: 13-43xc2x0 C.
(3) Formation of melanoid pigments:
(a) peptone/yeast/iron agar culture: none
(b) tyrosine agar culture: none
IV. Cell wall composition
In analysis for diaminopimelic acid by hydrolysis of the whole cells, only LL-diaminopimelic acid was detected.
The strain is classified in the genus Streptomyces of Actinomycetales, on the basis of the spore chains"" formation on the aerial mycelia and the configuration of diaminopimelic acid, etc.
Therefore, the strain was named as Streptomyces sp. RE-629, and has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, 1-3, Higashi 1 chome Tsukuba-ski Ibaraki-ken 305, Japan, under FERM BP-4126, as of Dec. 17, 1992. The deposited strain has been accepted for deposit under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purpose of Patent Procedure. All restrictions on the availability to the public of the deposited strain will be irrevocably removed upon the issuance of a patent.
For the culturing of the strains RE-701,and RE-629, a method usually used for the culturing of Actinomycetes is used. As the medium for culturing, either a natural medium or a synthetic medium can be used so long as it appropriately contains carbon sources, nitrogen sources, inorganic materials, etc. which may be assimilated by the cells.
As the carbon source, a carbohydrate such as glucose, fructose, sucrose, stabirose, starch, dextrine, mannose, maltose and molasses; an organic acid such as citric acid, malic acid, acetic acid and fumaric acid; an alcohol such as methanol and ethanol; a hydrocarbon such as methane, ethane, propane and n-paraffin; an amino acid such as glutamic acid; and glycerol can be used.
As the nitrogen source, an ammonium salt such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium phosphate, an amino acid such a aspartic acid, glutamine, cystine or alanine; and urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean powder, cottonseed lees, soybean casein, Casamino acid, pharmamedia (product of Procter and Gamble, U.S.A.), etc. can be used.
As the inorganic material, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium dihydrogen phosphate, magnesium sulfate, ferrous sulfate, manganese sulfate, copper sulfate, cobalt sulfate, zinc sulfate, calcium pantothenate, ammonium molybdate, aluminum potassium sulfate, barium carbonate, calcium carbonate, cobalt chloride, sodium chloride, etc. can be used.
In addition, vitamins such as thiamine, and other substances may be added as necessary to the culture medium in order to stimulate the production of Compound (I-1), Compound (I-2) or Compound (I-3).
When the microorganism utilized requires a specific substance, it is of course necessary to supplement the culture with the requisite substance.
Culturing is carried out by shaking culture, aeration stirring culture, etc. at 20-40xc2x0 C. and at a near neutral pH. Culturing is discontinued after 3 to 7 days, when the maximum amount of Compound (I-1), Compound (I-2) or Compound (I-3) is accumulated in the culture.
For isolation of the accumulated Compound (I-1), Compound (I-2) and Compound (I-3) from the culture, the ordinary method for isolating a physiologically active substance from cultures is used. Separation and collection of the Compound (I-1), Compound (I-2) and Compound (I-3) accumulated in the cells are carried out in a conventional manner as used for recovering the physiologically active substances from cells. For example, the cells are collected from the culture by filtration and centrifugation, and extracted with an organic solvent such as methanol, acetone, etc. Then the extract may be purified by partition chromatography, column chromatography or thin-layer chromatography using an adsorbent resin, silica gel, chemically modified silica gel, reverse phase silica gel, alumina, cellulose, diatomaceous earth, magnesium silicate, ion-exchange resin and the like, or gel filtration, to give Compound (I-1), Compound (I-2) and Compound (I-3).
In the above-described procedure, Compound (I-1), Compound (I-2) and Compound (I-3) may be detected by development on silica gel thin-layer chromatography, coloring by an iodine reaction or spraying of 50% sulfuric acid thereto, and heating. Alternatively, the detection is carried out by measurement of the absorption at a 253.7 nm wavelength with high performance liquid chromatography (hereunder referred to as HPLC) by use of a C-18 reverse phase silica gel column.
Compounds (I) and (II), including the substances Compound (I-1), Compound (I-2) and Compound (I-3) which are produced by culture of said microorganism, may be produced by synthetic means.
That is, Compounds (I) and (II) according to the present invention can be synthesized with a peptide synthesizer manufactured by Applied Biosystems, Inc., U.S.A. (ABI Co.) or manufactured by Shimadzu Seisakusho, using an Nxcex1-t-butyloxycarbonylamino acid or an Nxcex1-9-fluorenylmethyloxycarbonylamino acid whose side chains have been appropriately protected, following the synthesis programs of the same companies.
In addition, the cyclic peptide in Compound (I) may be obtained by synthesizing a partial peptide whose side chains have been appropriately protected, with the above-mentioned synthesizer or according to the usual liquid phase peptide synthesis method (xe2x80x9cFundamentals and Experiments in Peptide Synthesisxe2x80x9d, Izumiya, N. et al., Maruzen) described below, using a condensing agent such as benzotriazole-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate (PyBOP), etc. to obtain a cyclized partial peptide, and further using a peptide synthesizer or liquid phase synthesis method, or an appropriate combination of the two, condensing the C-terminal peptide with the cyclized partial peptide thus obtained. For the C-terminal peptide, Compound (II), for example, may be effectively used. 
The protected amino acid which is a starting material for Compounds (I) and (II) may be obtained from ABI Co., Shimadzu Seisakusho, Kokusan Chemicals, Inc., Nova Biochem Co. or Peptide Laboratories, Inc.
The thus obtained Compounds (I) and (II) may be purified by HPLC using a reverse phase column or any of the above mentioned chromatography methods.
A conventional method is used to obtain a pharmaceutically acceptable salt of Compound (I). That is, an acid addition salt or an organic base addition salt of Compound (I) can be obtained by dissolving Compound (I) in an aqueous solution of the corresponding acid or organic base, and freeze-drying the solution. Also, a metal salt of Compound (I) can be obtained by dissolving Compound (I) in an aqueous solution containing the corresponding metal ion, and purifying it by gel filtration of HPLC.
Embodiments of Compounds (I) and (II) are shown below in Table 1.
The pharmacological effects of representative compounds of Compound (I) are illustrated below, referring to Test Examples.